The present invention relates to isolated DNA molecules which encode the neuropeptide Y-Y5 receptor. In addition the present invention relates to the use of these molecules in the production of the neuropeptide Y-Y5 receptor using recombinant technology and to methods of screening and testing compounds for neuropeptide Y (NPY) agonist or antagonist activity.
In developed affluent countries the prevalence of obesity is alarming and it is now a massive contribution to morbidity and mortality in addition to being socially disadvantageous. Fat deposition in the abdominal area is a particular problem in relation to risk of Type II diabetes and cardiovascular disease. However, until recently, the molecular mechanisms controlling appetite, energy expenditure and adiposity have been surprisingly ill-understood.
Obesity has well-known associations with non-insulin-dependent diabetes (NIDDM), hypertension, dyslipidaemia and coronary heart disease, as well as less obvious links with diseases such as osteoarthritis and various malignancies; it also causes considerable problems through reduced mobility and decreased quality of life. Seven forms of rodent obesities, determined by single gene mutations, have been identified: yellow [Ay], adipose [Ad], diabetes [db], fat [fat], tubby [tub] and obese [ob] in the mouse and fatty [fa] in the rat. The obese phenotypes caused by these mutations differ in their age of onset, severity and the degree of insulin resistance. Similar phenotypes can also be seen in obese humans. Recently the molecular bases for some of these mutations has been elucidated. Of these the [ob] gene product xe2x80x9cleptinxe2x80x9d has created the most interest. However, many other factors are also involved in regulating energy balance and body fat distribution. Four factors appear most likely to have an important role: these are neuropeptide Y (NPY), corticotropin releasing factor (CRF)/ACTH/glucocorticoids, insulin and galanin. In particular, NPY and its receptors play an important role in the regulation of appetite and in a related manner, obesity.
Neuropeptide Y (NPY) forms a family (called the pancreatic polypeptide family) together with pancreatic polypeptide (PP) and peptide YY(PYY), which all consist of 36 amino acids and possess a common tertiary structure. Neuropeptide Y (NPY) receptors, members of the G protein-coupled receptor superfamily, are activated by one of the most abundant peptides in the mammalian nervous system and subsequently influence a diverse range of important physiological parameters, including effects on psychomotor activity, central endocrine secretion, anxiety, reproduction, vasoactive effects on the cardiovascular system and most importantly, potent effects on appetite. A number of neuropeptides and classical neurotransmitters, including noradrenaline and serotonin, modulate ingestive behaviours. However, NPY stands out from the many neurotransmitters with experimental effects on food intake in being able to induce obesity. Injections of NPY into the paraventricular nucleus (PVN), have been shown to increase, in a dose dependent manner, feeding and drinking behaviour in the rat. A single injection of NPY can increase food intake several-fold for several hours and is effective even during the light phase when rats usually eat little, and in animals that have already eaten to satiety. Consequently, NPY peptides are certainly among the most potent orexygenic substances known in either food deprived or satiated animals. Repeated NPY injections into the PVN result in a massive and persistent feeding response and the rats ultimately develop obesity, with a true increase in body fat content. The importance of NPY as a mediator of appetite/obesity regulation is further enhanced by the very recent report that the obese gene product leptin inhibits NPY synthesis and release.
Injections of NPY into the paraventricular nucleus cause a prompt and robust increase in plasma ACTH levels and there is clear evidence that NPY-induced ACTH secretion is mediated by corticotropin releasing factor (CRF). However, its mode of action as well as its interaction with CRF within the brain is largely unknown, as are its interrelationships with other hormones, such as insulin. Nevertheless an agent which increases appetite and raises glucocorticoid levels might be important in generating central obesity.
Specific agonists and antagonists of NPY are therefore likely to be of substantial benefit for therapy of a wide range of clinical disorders. As NPY possess a compact tertiary structure and different parts of the molecule are required for interaction with different subtypes of the receptor, the logical developments of both agonists and antagonists is critically dependent upon the availability and knowledge of specific receptor structure.
It is presently known that NPY binds specifically to at least five receptors; Y1, Y2, Y3, Y4 and Y1-like (or xe2x80x9catypical Y1xe2x80x9d). While it has been demonstrated that NPY receptors couple to the adenylate cyclase second messenger system, it remains probable that additional NPY receptor subtypes exist since there is evidence that phosphatidylinositol turnover, cations, and arachidonic acid may also function as second messengers for NPY.
Since NPY agonists and antagonists may have commercial value as, for example, potential anti-hypertensive agents, cardiovascular drugs, neuronal growth factors, anti-psychotics, anti-obesity and anti-diabetic agents, the ability to produce NPY receptors by recombinant DNA technology would be advantageous. To this end, DNA molecules encoding Y1, Y2, Y3 and Y4 have previously been isolated.
The present inventors have now isolated novel DNA molecules encoding the human, mouse and rat Y1-like (hereinafter referred to as NPY-Y5) receptors. Similar DNA molecules encoding human and rat NPY-Y5 have been described in International (PCT) Patent Specification No. WO 96/16542, however, these encode receptors with, in the case of the human NPY-Y5, an additional 10 N-terminus amino acids, and, in the case of the rat 20 NPY-Y5, an additional 11 N-terminus amino acids. Through analysis of several cDNA clones and RT-PCR using specific primers for intron and exon sequences, the present inventors have confirmed that the human, mouse and rat NPY-Y5 receptor does not include these additional 10/11 amino acids. The DNA molecules described in WO 96/16542 may thus exhibit lower expression rates over those of the present invention. In addition, the receptors encoded by the DNA molecules described in WO 96/16542, may show lower and possibly altered activity.
Thus, in a first aspect, the present invention provides an isolated DNA molecule encoding an NPY-Y5 receptor having about 445 amino acids or a functionally equivalent fragment thereof.
Preferably, the isolated DNA molecule encodes an human, mouse or rat NPY-Y5 receptor.
Most preferably, the isolated DNA molecule has a nucleotide sequence substantially corresponding or, at least,  greater than 80% (more preferably,  greater than 95%) homologous to that shown:
(i) at nucleotides 6291 to 7625 of FIG. 1 (SEQ ID NO:1),
(ii) at nucleotides 63 to 1397 of FIG. 2 (SEQ ID NO:3),
(iii) at nucleotides 115 to 1449 of FIG. 3 (SEQ ID NO:5), or
(iv) at nucleotides 73 to 1470 of FIG. 4 (SEQ ID NO:7).
The isolated DNA molecule may be incorporated into plasmids or expression vectors, which may then be introduced into suitable bacterial, yeast and mammalian host cells. Such host cells may be used to express the NPY-Y5 receptor encoded by the isolated DNA molecule.
Accordingly, in a second aspect, the present invention provides a mammalian, yeast or bacterial host cell transformed with the DNA molecule of the first aspect.
In a third aspect, the present invention provides a method of producing NPY-Y5 receptors comprising culturing the host cell of the second aspect under conditions enabling the expression of the DNA molecule and optionally recovering the NPY-Y5 receptor.
Preferably, the host cell is mammalian or bacterial. Where the cell is mammalian, it is presently preferred that it be a Chinese hamster ovary (CHO) cell, human embryonic kidney 293 cell or insect Sf9 cells.
In a preferred embodiment, the NPY-Y5 receptor is expressed onto the surface of the host cell.
The DNA molecules of the present invention represent a NPY receptor which may be of interest both clinically and commercially as it is expressed in many regions of the body and NPY affects a wide number of systems.
By using the nucleic acid molecules of the present invention it is possible to obtain neuropeptide Y-Y5 receptor protein in a substantially pure form.
Accordingly, in a fourth aspect, the present invention provides NPY-Y5 receptor in a substantially pure form.
Preferably, the purified NPY-Y5 has an amino acid sequence substantially corresponding to any one of the amino acid sequences shown in FIG. 5.
In a fifth aspect, the present invention provides an antibody capable of specifically binding to an NPY-Y5 receptor.
In a sixth aspect, the present invention provides a non-human animal transformed with a DNA molecule according to the first aspect of the present invention.
In a seventh aspect, the present invention provides a method for detecting agonist or antagonist agents of NPY-Y5 receptor, comprising contacting a NPY-Y5 receptor or a cell transfected with and expressing the DNA molecule of the first aspect with a test agent under conditions enabling the activation of a NPY-Y5 receptor, and detecting an increase or decrease in NPY-Y5 receptor activity.
In a further aspect, the present invention provides a nucleic acid probe comprising a nucleotide sequence of 10 or more nucleotides capable of specifically hybridising to a unique sequence within the DNA molecule of the first aspect.
In a still further aspect, the present invention provides an antisense nucleic acid molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes NPY-Y5 receptor so as to prevent translation of the mRNA molecule. Such antisense nucleic acid molecules may include a ribozyme region to catalytically inactivate mRNA to which it is hybridised.
The term xe2x80x9csubstantially correspondingxe2x80x9d as used herein in relation to the nucleotide sequences shown in FIGS. 1 and 2 is intended to encompass minor variations in the nucleotide sequence which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.
The term xe2x80x9csubstantially correspondingxe2x80x9d as used herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity the NPY-Y5 receptor. These variations may include conservative amino Aid substitutions. The substitutions envisaged are:
G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nxcex1-alkalamino acids.
The invention is hereinafter described by way of the following non-limiting example and further, with reference to the accompanying figures.